Question: Featurecounts to DESEq2 - How to merge biological replicates?
0
gravatar for Sreeraj Thamban
2.4 years ago by
Indian Institute of Science Education and Research
Sreeraj Thamban140 wrote:

Hi I have four aligned BAM files for two conditions, treated and untreated,each of them having two biological replicates, I could run featurecounts for the four BAM files separtly,but I dont know how to make these output files compatible for downstream differential gene expression analysis using DESeq2, can anyone help please... Thank you.

rna-seq featurecounts deseq2 • 2.1k views
ADD COMMENTlink written 2.4 years ago by Sreeraj Thamban140
2

Everything is explained in DESeq2 manual.

1) Get the count (featurecount is a good tool for that) 2) Provide 3 variables: a. A matrix of the count (rows=genes, column=samples). b. A matrix with all the informations on your samples (names, group=control/case etc) c. A design (Ex: ~group) 3) Run DESeq2 following the manual

Regarding the output of featureCount you should get for each sample a dataframe containing by row the genes (and info) with the last column being the count associated to each gene. You can simply combine the outputs by column to get the matrix of count require by DESeq.

ADD REPLYlink written 2.4 years ago by VHahaut1.1k
1

featureCounts produces a matrix directly when you feed it multiple BAM files (provide them in the order you want the samples to be in the matrix columns).

ADD REPLYlink written 2.4 years ago by genomax71k

Dear Genomax, can you tell me how to feed multiple bam files to featurecounts?

ADD REPLYlink written 2.4 years ago by Sreeraj Thamban140
1

featureCounts [options] -o counts.txt file1.bam file2.bam file3.bam etc

ADD REPLYlink modified 2.4 years ago • written 2.4 years ago by genomax71k
1

You need a matrix file with genes as your rows and samples as your columns. No need to merge the replicates.

ADD REPLYlink written 2.4 years ago by genomax71k

Hi Genomax2, I have the matrix files for all bam files, but how to load these files to Deseq2? I have been using kallisto - tximport-deseq2 pipeline and there it was straight forward, Thanks

ADD REPLYlink written 2.4 years ago by Sreeraj Thamban140

help(read.delim)

ADD REPLYlink written 2.4 years ago by Devon Ryan92k
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